Materials Science and Technology of Polymers at the University of Twente

Chemistry in block copolymer nanocontainers
Self-assembly, container properties and confined enzymatic reactions

The thesis describes thoroughly the preparation and characterization of vesicles made from amphiphilic block copolymers and subsequently their use as nanocontainers for the study of enzymatic reactions in atto-liter confined space. The confinement caused by the membrane boundaries of the vesicles was found to enhance the catalytic activity of the encapsulated enzymes. This enhancement was attributed to a combined effect of high collision frequencies between the reagents and a strong interaction between the enzymes and the vesicle wall, which was predicted in previous theoretical studies yet not observed experimentally. In addition to kinetic investigation of enzymatic catalysis, the “polymersome” nanocontainers were characterized and their thermodynamics in mixed solvents was studied.
The preparation of vesicles made from polystyrene-block-poly (acrylic acid) (PS-b-PAA) using the selective solvent method is described in Chapter 3. Solution composition, including the water content, initial polymer concentration, added ions and the nature of the common solvent were varied to control the size of the vesicles. The effect of the chain length of the block copolymers on the wall thickness of the vesicles was explored. Preparation of vesicles at different temperatures was carried out and the morphological change from vesicles to micelles at elevated temperature was observed. The thermal stability of the vesicles was studied using atomic force microscopy (AFM) and it was found that substrate supported vesicles were stable in air up to 80C. Finally small fluorescent molecules with different hydrophobicities were encapsulated both in the wall and in the interior of the vesicles as a first attempt to utilize these vesicles as nanocontainers for future study of biochemical reactions in confined environment.
Chapter 4 describes the assessment of the mechanical properties of PS-b-PAA vesicles assembled from polymers with different chain lengths, i.e. different membrane thickness, using AFM. The vesicle membrane thickness was shown by transmission electron microscopy (TEM) to scale in accordance with literature data with the degree of polymerization of both blocks. Based on AFM force data and the application of the shell deformation theory, the apparent isotropic Young’s moduli of the membranes were estimated. While the values of the spring constant of the membrane were found to be directly proportional to the membrane thickness for vesicles with the same diameter, the apparent Young’s moduli were found to decrease with increasing wall thickness. This effect coincides with the decreasing degree of polystyrene stretching that was reported in the literature for membranes with increasing thickness.
In Chapter 5 the study of loading enzyme trypsin and its fluorogenic substrate R110-Arg2 into the vesicles is discussed. The catalytic turnover number (Kcat) and the Michaelis-Menten constant (Km) for the unrestricted reaction of trypsin in solution and for the reaction confined in the vesicles were found to differ significantly. While the values of Kcat of trypsin encapsulated in vesicles with different diameters were always higher than those of trypsin in solution, the values of Km were lower. Compared to the reaction in solution, the enzyme efficiency (Kcat/Km) increased by two orders of magnitude. This observed higher reactivity of encapsulated trypsin is attributed to the molecular confinement inside the vesicles, which causes higher collision frequencies between the reagents as well as higher collision frequencies between the encapsulated molecules and the container wall.
The study of enzymatic reactions in nanocontainers and the investigation of confinement effects on the catalytic activity of enzymes are extended and present in Chapter 6. a-Chymotrypsin and its fluorogenic substrate N-succinyl-Ala-Ala-Phe- 7-amido-4-methylcoumarin were encapsulated into PS-b-PAA vesicles with different sizes. The encapsulation percentages of the enzymes and substrates were found to be the same when loaded into vesicles with the same size. The efficiency of the encapsulation increased as the size of the vesicles decreased. The values of Kcat of the enzymatic reactions encapsulated in vesicles with different diameters were always higher (15 times for the most pronounced enhancement) than those of the enzymatic reactions in solution. A clear size dependence of the Kcat of enzymatic reactions inside the capsules was observed for vesicles with an inner diameter ranging from 30 nm to 170 nm. The observed higher reactivity of encapsulated a-chymotrypsin is attributed to the molecular confinement inside the vesicles, which is a combined effect of high collision frequencies between the reagents and a significant interaction between the enzymes and the vesicle wall.
The study on the size and morphology of PS-b-PAA aggregates in a (THF)/H2O mixed solvent system at different temperatures is presented in Chapter7. It was found that PS-b-PAA aggregates underwent a vesicle-to-micelle transition as the temperature of the system was raised above a critical value. The transition was a result of the change in polymer solvent interaction at different temperatures. In addition, the presence of the common solvent THF was found to be crucial for the transition, as this lowered the glass transition temperature of polystyrene in the vesicle membrane to allow the polymer chains to reorganize. Successful encapsulation and release of small molecules was achieved utilizing the assembly and disassembly of PS-b-PAA aggregates cross the transition temperature. Rhodamine 110 bisamide, whose hydrolysis reaction was discussed in Chapter 5, was preloaded into the vesicles and released when the system was heated to above the transition temperature to react with enzyme trypsin to form fluorescent product rhodamine 110. The product was then encapsulated into reformed vesicles when the system was cooled down to below transition temperature, proven using sodium nitrite.
Finally, the results of a study on the immobilization and patterning of PS-b-PAA vesicles onto amino-functionalized solid supports via electrostatic interactions is discussed in Chapter8. The electrostatic nature of the immobilization of the vesicles on surfaces was confirmed by varying the pH of the solution during the immobilization experiments. Vesicles were found to have a higher affinity to the surface at neutral pH as electrostatic attraction was minimized at both low and high pH. The ionic strength was also used to control the amount of vesicles immobilized on the surface. A higher salt concentration resulted in an increased number of vesicles deposited due to the screening of the electrostatic repulsion. Micromolding in capillaries (MIMIC) was used in combination with the electrostatic immobilization to create line patterns of PS-b-PAA vesicles containing trypsin and a fluorogenic substrate. This approach may serve as a potential means to fabricate microarray systems and may find future applications in real-time observations of reaction kinetics and dynamic behavior of biomolecules of interest inside the vesicles.